N-benzyl anthracyclines

ABSTRACT

N-Benzyl and N,N-dibenzyl derivatives of the anthracycline compounds adriamycin and daunomycin, said derivatives having anticancer properties and being active against leukemia P-388 in mice, for example. The N,N-dibenzyl derivatives have the unique quality of exhibiting a loss of DNA-binding properties.

ORIGIN OF INVENTION

The invention described herein was made in the course of or under a contract with the U.S. Department of Health, Education, and Welfare.

This application is a continuation-in-part of Ser. No. 842,787 filed Oct. 17, 1977 now abandoned.

SUMMARY OF INVENTION

The present invention relates to the provision of novel N-benzyl and N,N-dibenzyl derivatives of adriamycin and daunomycin and to compositions containing the same, said derivatives having utility as anticancer chemicals and exhibiting the structure ##STR1## wherein R represents --COCH₃, --CHOH--CH₃, --COCH₂ OH or --CHOH--CH₂ OH and wherein X represents --NHCH₂ Ph or --N(CH₂ Ph)₂ in which "Ph" represents a phenyl group, and their pharmaceutically acceptable acid addition salts.

For ease of identification, the eight compounds falling within the scope of the present invention are enumerated (I through VIII) in Table 1 below.

                  Table 1                                                          ______________________________________                                                    X = NHCH.sub.2 Ph                                                                          X = N(CH.sub.2 Ph).sub.2                                ______________________________________                                         R = COCH.sub.3                                                                              I             V                                                   R = CHOC--CH.sub.3                                                                          II            VI                                                  R = COCH.sub.2 OH                                                                           III           VII                                                 R = CHOH--CH.sub.2 OH                                                                       IV            VIII                                                ______________________________________                                    

The preparation of the foregoing compounds is described in detail by the examples. However, it may be noted here that reductive alkylation of daunomycin (IX) with benzaldehyde in the presence of sodium cyanoborohydride for prolonged reaction periods at room temperature produces good yields of the N,N-dibenzyl compound (V), plus the 13-dihydro derivative (VI) as byproduct. With shorter reaction periods, the mono-N-benzyldaunomycins I and II predominate. Either N-benzylation or N,N-dibenzylation can be favored by choosing the stoichiometry. N,N-Dibenzyladriamycin (VII) was similarly obtained by treating adriamycin (X) with benzylaldehyde and sodium cyanoborohydride under conditions that gave predominantly mono-N-benzylation, compound (III). Since reductive alkylation is accompanied by ketone reduction, four products (I, II, V, VI) were observed in the benzylation of daunomycin and four more (III, IV, VII, VIII) from the benzylation of adriamycin. Compound VIII, though identified in analytical procedures, was not isolated. However, it is believed to have anticancer activity paralleling that of the other compounds of this invention.

The compounds of the present invention can be prepared either in the free base or acid addition salt form. The salts are soluble in water and aqueous propylene glycol, for example, while the compounds in free base form are soluble in selected organic solvents such as chloroform, methylene chloride and ethyl acetate, for example. The salts are thus particularly well adapted for use in antitumor applications since they may be used in aqueous (including saline) solution form. These acid addition salts (prepared here as those of HCl) are preferably the pharmaceutically acceptable, nontoxic addition salts with suitable acids such as those with inorganic acids, for example, hydrochloric, hydrobromic, nitric, sulphuric and phosphoric acids, and with organic acids, such as organic carboxylic acids, for example, glycolic, maleic, hydroxymaleic, malic, tartaric, citric, salicylic acids, and organic sulphonic acids, for example, methanesulphonic and toluene-p-sulphonic acids.

An acid addition salt can be converted into the free compound according to known methods, for example, by treating it with a base, such as with a metal hydroxide or alkoxide, for example, an alkali metal or alkaline earth metal hydroxide, for example, lithium hydroxide, sodium hydroxide, potassium hydroxide or calcium hydroxide; with a metal carbonate, such as an alkali metal or an alkaline earth metal carbonate or hydrogen carbonate, for example sodium, potassium or calcium carbonate or hydrogen carbonate; with ammonia; or with a hydroxyl ion exchange resin, or with any other suitable reagent.

An acid addition salt may also be converted into another acid addition salt according to known methods; for example, a salt with an inorganic acid may be treated with a metal salt, for example a sodium, barium or silver salt, of an acid in a suitable diluent, in which a resulting inorganic salt is insoluble and is thus removed from the reaction medium. An acid addition salt may also be converted into another acid addition salt by treatment with an anion exchange preparation.

The preparation of each of the foregoing compounds is described in the following examples:

EXAMPLE 1 N-Benzyldaunomycin (I), N-Benzyl-13-dihydrodaunomycin (II) and N,N-Dibenzyldaunomycin (V), and HCl salts

Daunomycin.HCl (2.26 g, 4.0 mmol) and benzaldehyde (8.2 mL, 80.0 mmol) were placed in 3:1 acetonitrile/H₂ O (116 mL) and stirred at 23° for 0.5 hour. NaCNBH₃ (0.75 g, 12.0 mmol) was added and stirring was continued for 0.5 hour. The reaction was diluted with H₂ O (150 mL) and extracted with CHCl₃ (3×100 mL). The extracts were combined, washed with H₂ O (30 mL) and the aqueous phase reextracted with CHCl₃ (50 mL). The organic solutions were combined and extracted with cold 0.1 N HOAc (6×100 mL). The extracts were combined, immediately basified with NaHCO₃ and extracted with CHCl₃ (3×300 mL). The extracts were combined, evaporated and the residue chromatographed (1.3 Kg silica gel--50 mm×7 ft dry column--10:1 CHCl₃ /MeOH) to afford 1.06 g of N-benzyldaunomycin (I) (51%) and 0.17 g of N-benzyl-13-dihydrodaunomycin (II) (7%) as the free bases. The organic solution after the HOAc extraction was dried and evaporated. The residue was chromatographed (560 g silica gel--50 mm×3 ft dry column--10:1 CHCl₃ /MeOH) to afford 0.18 g (6%) of N,N-dibenzyldaunomycin (V) free base. The HCl salts were prepared by reacting a CHCl₃ solution of each compound with an equivalent amount of methanolic HCl followed by precipitation with ether to afford; 1.28 g (49%) of (I); [α]_(D) +252° (c 0.05 95% EtOH); tlc (10:1 CHCl₃ /MeOH) R_(f) 0.60,

    ______________________________________                                         Anal. calcd for C.sub.34 H.sub.35 NO.sub.10 . HCl . H.sub.2 0:                        C       H         N         Cl                                          ______________________________________                                                  60.76     5.70      2.08    5.28                                      Found:   60.88     5.59      1.99    5.40;                                     ______________________________________                                    

0.16 g (6%) of (II); [α]_(D) +207°, (c 0.047, 95% EtOH); tlc (10:1 CHCL₃ /MeOH) R_(f) 0.4,

    ______________________________________                                         Anal. calcd for C.sub.34 H.sub.37 NO.sub.10 . HCl . 1.25 H.sub.2 O:                   C       H         N         Cl                                          ______________________________________                                                  60.17     6.02      2.06    5.22                                      Found:   60.07     5.79      2.26    5.38;                                     ______________________________________                                    

and 0.16 g (5%) of (V); [α]_(D) +283° (c 0.048, 95% EtOH); tlc (10:1 CHCl₃ /MeOH) R_(f) 0.90,

    ______________________________________                                         Anal. calcd for C.sub.41 H.sub.41 NO.sub.10 . HCl . 0.75 H.sub.2 O:                   C       H         N         Cl                                          ______________________________________                                                  64.99     5.79      1.85    4.68                                      Found:   65.08     5.69      1.77    4.45.                                     ______________________________________                                    

EXAMPLE 2 N-Benzyladriamycin (III), N,N-Dibenzyladriamycin (VII), and N-Benzyl-13-dihydroadriamycin (IV), and HCl salts

Adriamycin.HCl (2.90 g, 5.0 mmol) and benzaldehyde (10.5 mL, 100 mmol) were placed in 2:1 acetonitrile/H₂ O (150 mL) and stirred at 23° for 20 minutes. The solution was added dropwise over 15 minutes to a solution of NaCNBH₃ (0.94 g, 15.0 mmol) and benzaldehyde (10.5 mL) in 2:1 acetonitrile/H₂ O (60 mL). Stirring was continued for 30 minutes and the reaction was diluted with H₂ O (150 mL) and extracted with CHCl₃ (4×100 mL). The extracts were combined, washed with saturated NaCl (2×100 mL), dried and evaporated. The residue was chromatographed (1.3 kg silica gel--50 mm×7 ft dry column--40:10:1 CHCl₃ /MeOH/H₂ O) to afford 0.10 g (3%) N,N-dibenzyladriamycin (VII), 1.02 g (30%) of N-benzyladriamycin (III), and 1.20 g (38%) of N-benzyl-13-dihydroadriamycin (IV) as the free bases. Eluting after (VII) was a small amount of material, never completely purified, but tentatively identified as N,N-dibenzyl-13-dihydroadriamycin (VIII). These were converted to the HCl salts by the procedure described in the previous example to afford 0.07 g (2%) of (VII); [α]_(D) +275° (c 0.051, 85% EtOH); tlc (10:1 CHCl₃ /MeOH) R_(f) 0.85,

    ______________________________________                                         Anal. calcd for C.sub.41 H.sub.41 NO.sub.11 . HCl . 0.6 H.sub.2 O:                    C       H         N         Cl                                          ______________________________________                                                  63.86     5.64      1.81    4.59                                      Found:   63.88     5.57      1.83    4.45;                                     ______________________________________                                    

0.90 g (28%) of (III); [α]_(D) +209° (c. 0.048, 95% EtOH); tlc (10:1 CHCl₃ /MeOH) R_(f) 0.55,

    ______________________________________                                         Anal. calcd for C.sub.34 H.sub.35 NO.sub.11 . HCl . H.sub.2 O:                        C       H         N         Cl                                          ______________________________________                                                  59.34     5.56      2.03    5.15                                      Found:   59.46     5.40      2.01    5.37;                                     ______________________________________                                    

and 1.00 g (30%) of (IV); [α]_(D) +204° (c 0.050, 95% EtOH); tlc (10:1 CHCl₃ /MeOH) R_(f) 0.25,

    ______________________________________                                         Anal. calcd for C.sub.34 H.sub.37 NO.sub.11 . HCl . 0.8 H.sub.2 O:                    C       H         N         Cl                                          ______________________________________                                                  59.48     5.87      2.04    5.16                                      Found:   59.43     5.81      2.05    5.40.                                     ______________________________________                                    

EXAMPLE 3 N,N-Dibenzyldaunomycin (V), N,N-Dibenzyl-13-dihydrodaunomycin (VI), N-Benzyldaunomycin (I) and N-Benzyl-13-dihydrodaunomycin (II), and HCl salts

Daunomycin.HCl (2.54 g, 4.5 mmol) and benzaldehyde (9.2 mL, 90.0 mmol) were placed in 2:1 acetonitrile/H₂ O (90 mL) and stirred at 23° for 20 minutes. The solution was added dropwise over 20 minutes to a stirred solution of NaCNBH₃ (0.57 g, 9.0 mmol) in acetonitrile (20 mL) and stirring was continued for 14 days. The reaction was diluted with H₂ O (150 mL) and extracted with CHCl₃ (5×100 mL). The extracts were combined, washed with H₂ O (100 mL) and the aqueous layer extracted with CHCl₃ (50 mL). The organic solutions were combined, dried and evaporated and residue chromatographed (190 g silica gel--2.8×64 cm column --CHCl₃ to 9:1 CHCl₃ /MeOH) to afford in order of elution 1.19 g (38%) of (V), 0.41 g (13%) of N,N-dibenzyl-13-dihydrodaunomycin (VI), 0.30 g (11%) of (I) and 0.10 g (4%) of (II) as the free bases. These were converted to HCl salts by the above procedure to afford 1.20 g (35%) of (V) and 0.37 (11%) of (VI); [α]_(D) +247° (c 0.049, 95% EtOH), tlc (10:1 CHCl₃ /MeOH) R_(f) 0.85.

    ______________________________________                                         Anal. calcd for C.sub.41 H.sub.43 NO.sub.10 . HCl . 1.5 H.sub.2 O:                    C       H         N         Cl                                          ______________________________________                                                  63.68     6.13      1.81    4.58                                      Found:   63.66     5.75      1.78    4.86;                                     ______________________________________                                    

and 0.23 g (8%) of (I) and 0.07 g (2%) of (II), as HCl salts.

The compounds of this invention, including the salts thereof, can be administered by any available route, including oral and parenteral (intravenous, intraperitoneal, subcutaneous, and intramuscular) administration. The amount administered is sufficient to ameliorate the leukemia or other type of cancer against which the compounds hereof may prove to be effective. For example, in the treatment of lower test animals, a dosage of a compound of the present invention within the range from about 0.1 mg/kg to about 500 mg/kg per day should be sufficient to ameliorate leukemia. The upper dosage limit is that imposed by toxic side effects and can be determined by trial and error for the animal to be treated.

To facilitate administration, the compounds of this invention, including the salts thereof, can be provided in composition form, and preferably in dosage unit form. While any compound selected can be administered per se, it is normally administered in conjunction with a pharmaceutically acceptable carrier therefor, which dilutes the compound and facilitates handling. The term "pharmaceutically acceptable" means that the carrier (as well as the resulting composition) is sterile and nontoxic.

The carrier or diluent can be solid, semisolid, or liquid, and can serve as a vehicle, excipient, or medium for the anti-cancer agent. Exemplary diluents and carriers include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, mineral oil, cocoa butter, oil of theobroma, alginates, tragacanth, gelatin, syrup, methyl cellulose, polyoxyethylene sorbitan, monolaurate, methyl- and propyl-hydroxybenzoate, talc or magnesium stearate.

For convenience in handling, the compounds hereof and the carrier or diluent can be enclosed or encapsulated in a capsule, sachet, cachet, gelatin, paper or other container, especially when intended for use in dosage units. The dosage units can for example take the form of tablets, capsules, suppositories or cachets.

The following examples illustrate various forms of dosage units in which the HCl salt of compound V can be prepared.

EXAMPLE 4

    ______________________________________                                         Tablet formulation   Mg/tablet                                                 ______________________________________                                         Compound V           15                                                        Lactose              86                                                        Cornstarch (dried)   45.5                                                      Gelatin              2.5                                                       Magnesium stearate   1.0                                                       ______________________________________                                    

Compound V is powdered and passed through a mesh sieve and well mixed with the lactose and 30 mg of the cornstarch, both passed through a sieve.

The mixed powders are massed with a warm gelatin solution, prepared by stirring the gelatin in water and heating to form a 10% w/w solution. The mass is granulated by passing through a sieve, and the moist granules dried at 40° C.

The dried granules are regranulated by passing through a sieve and the balance of the starch and the magnesium stearate is added and thoroughly mixed.

The granules are compressed to produce tablets each weighing 150 mg.

EXAMPLE 5

    ______________________________________                                         Tablet formulation   Mg/tablet                                                 ______________________________________                                         Compound V           100                                                       Lactose              39                                                        Cornstarch (dried)   80                                                        Gelatin              4.0                                                       Magnesium stearate   2.0                                                       ______________________________________                                    

The method of preparation is identical with that of Example 4 except that 60 mg of starch is used in the granulation process and 20 mg during tableting.

EXAMPLE 6

    ______________________________________                                         Capsule formulation  Mg/capsule                                                ______________________________________                                         Compound V           250                                                       Lactose              150                                                       ______________________________________                                    

Compound V and lactose are passed through a sieve and the powders well mixed together before filling into hard gelatin capsules of suitable size, so that each capsule contains 400 mg of mixed powders.

EXAMPLE 7

    ______________________________________                                         Suppositories       Mg/suppository                                             ______________________________________                                         Compound V           50                                                        Oil of Theobroma    950                                                        ______________________________________                                    

Compound V is powdered and passed through a sieve and triturated with molten oil of theobroma at 45° C. to form a smooth suspension.

The mixture is well stirred and poured into molds, each of nominal 1 g capacity, to produce suppositories.

EXAMPLE 8

    ______________________________________                                         Cachets              Mg/cachet                                                 ______________________________________                                         Compound V           100                                                       Lactose              400                                                       ______________________________________                                    

Compound V is passed through a mesh sieve, mixed with lactose previously sieved and fitted into cachets of suitable size so that each contains 500 mg.

EXAMPLE 9

    ______________________________________                                         Intramuscular injection                                                        (sterile suspension in                                                         aqueous vehicle)       Mg                                                      ______________________________________                                         Compound V             10                                                      Sodium citrate         5.7                                                     Sodium carboxymethylcellulose                                                  (low viscosity grade)  2.0                                                     Methyl para-hydroxybenzoate                                                                           1.5                                                     Propyl para-hydroxybenzoate                                                                           0.2                                                     Water for injection to 1.0 ml                                                  ______________________________________                                    

EXAMPLE 10

    ______________________________________                                         Intraperitoneal, intravenous                                                   or subcutaneous injection                                                      (sterile solution in aqueous                                                   carrier system)        Mg                                                      ______________________________________                                         Compound V, hydrochloric acid                                                  addition salt          15                                                      Sodium citrate         5.7                                                     Sodium carboxymethylcellulose                                                  (low viscosity grade)  2.0                                                     Methyl para-hydroxybenzoate                                                                           1.5                                                     Propyl para-hydroxybenzoate                                                                           0.2                                                     Water for injection to 1.0 ml                                                  ______________________________________                                    

The other compounds of this invention could be prepared in dosage unit form in the same general fashion as that described above for compound V.

BIOLOGICAL TESTS

Sufficient samples of all but compound (VIII) have been obtained for biological screening, and recent results of tests both in vitro and in vivo are shown in Table 2 below. It will be seen that every compound was active against leukemia P388 in mice, with N,N-dibenzyldaunomycin (compound VI) being clearly the best. The in vitro results are surprising and appear to be highly significant in that there is an abrupt and unexpected loss of DNA-binding properties in going from N-benzyl to N,N-dibenzyl compounds. ΔT_(m) is the most direct measure of DNA-binding but the inhibition of nucleic acid synthesis is also lost. Most of the anthracyclines bind to DNA, presumably by an intercalation mechanism and are mutagenic. In these N,N-dibenzyl compounds the anti-tumor activity in the mouse screen is retained but the DNA-binding properties are lost. This suggests the N,N-dibenzyl compounds may be useful and effective anti-cancer agents, but may act by a different mechanism from most of the other anthracyclines, and thus may avoid the serious cardiotoxic side effects common to the anthracyclines now in use.

The compound referred to herein as daunomycin (IX) is otherwise known as daunorubicin or rubidomycin.

                                      Table 2                                      __________________________________________________________________________     IN VITRO AND IN VIVO TESTS OF N-BENZYL ANTHRACYCLINES                                                      L1210 Cells.sup.c                                                              Inhib of Synth                                                                              Highest Mean T/C vs P388 in                                                    Mice.sup.d                            Compound               ΔTm                                                                           DNA    RNA   qd 1-9     q4d 5,9,13                 No.   NSC No..sup.a    (° C.).sup.b                                                                 ED.sub.50, μM                                                                      ED.sub.50, μM                                                                     % T/C (dose, mg/kg)                                                                       % T/C (dose,               __________________________________________________________________________                                                         mg/kg)                           Parents                                                                  IX    82151 Daunomycin 11.2 1.0    0.3   167 (0.5)  134 (8)                    X     123127 Adriamycin                                                                               13.4 1.5    0.67  193 (1.0)  157 (8)                          N-Benzyl                                                                 I     268241 Dnm, N-benzyl                                                                            10.2 1.6    0.17  187 (6.25) 226 (18.8)                 II    268240 Dnm, N-benzyl, 13-diH                                                                    6.25 1.7    0.32  226 (6.25) --                         III   269433 Adm, N-benzyl                                                                            11.3 0.65   0.09  224 (6.25) 200 (18.8)                 IV    269434 Adm, N-benzyl, 13-diH                                                                    7.6  1.4    0.29  196 (1.56) 143 (37.5)                       N,N-Dibenzyl                                                             V     268242 Dnm, N,N-dibenzyl                                                                        1.35 >100   10    259 (25)   227 (37.5)                 VI    278170 Dnm, N,N-dibenzyl, 13-diH                                                                0.6    220 (susp)                                                                          7.6   229 (40)   233 (50)                   VII   269435 Adm, N,N-dibenzyl                                                                        -1.2 110    4.8   175 (6.25) --                         __________________________________________________________________________      .sup.a NSC accession number of the National Cancer Institute.                  .sup.b ΔT.sub.m values represent the difference in temperature           between that at which the DNA denatures (or melts) without the drug and        that at which denaturing occurs in the presence of the drug;                   ΔT.sub.m values around 1° suggest weak binding, if any, to        isolated helical DNA; values ≧5° suggest a significant           degree of binding.                                                             .sup.c Assay described in G. Tong, W. W. Lee, D. R. Black and D. W. Henry      J. Med. Chem., 19, 395 (1976).                                                 .sup.d Ip P388 leukemia treated ip on QD1-9 and Q4D 5,9,13 schedules           according to standard NCI protocols. Assay described in R. I. Geran, N. H      Greenberg, M. M. MacDonald, A. M. Schumacker and B. J. Abbott, Cancer          Chemother. Rep., Part 3, 3, (No. 2), 9 (1972), Protocol 1,200. T/C = rati      of survival time of treated mice to that of untreated controls times 100.      Untreated controls survive about 9 days. For Dnm and Adm, % T/C values ar      means from several dozen tests, with standard deviation up to ±40. For      the Nbenzyl compounds, % T/C values are means from all the tests (1 to 3)      so far, at the optimum dose.                                              

What is claimed is:
 1. Compounds having the structure ##STR2## wherein R represents --COCH₃, --CHOH--CH₃, --COCH₂ OH and --CHOH--CH₂ OH and wherein X represents --NHCH₂ Ph and N(CH₂ Ph)₂, and their pharmaceutically acceptable acid addition salts, with the provision that where R is --CHOH--CH₂ OH, X is not N(CH₂ Ph)₂.
 2. The compound of claim 1 which is N-benzyldaunomycin, together with its pharmaceutically acceptable acid addition salts.
 3. The compound of claim 1 which is N-benzyl-13-dihydrodaunomycin, together with its pharmaceutically acceptable acid addition salts.
 4. The compound of claim 1 which is N-benzyladriamycin, together with its pharmaceutically acceptable acid addition salts.
 5. The compound of claim 1 which is N-benzyl-13-dihydroadriamycin, together with its pharmaceutically acceptable acid addition salts.
 6. The compound of claim 1 which is N,N-dibenzyldaunomycin, together with its pharmaceutically acceptable acid addition salts.
 7. The compound of claim 1 which is N,N-dibenzyl-13-dihydrodaunomycin, together with its pharmaceutically acceptable acid addition salts.
 8. The compound of claim 1 which is N,N-dibenzyladriamycin, together with its pharmaceutically acceptable acid addition salts.
 9. A dosage unit of a pharmaceutical composition for treating leukemia L1210 and P388 in mice comprising an amount within the range of from about 0.1 to about 500 mg per dosage unit, therapeutically effective to ameliorate leukemia, of at least one compound selected from the group consisting of N-benzyldaunomycin, N-benzyl-13-dihydrodaunomycin, N-benzyladriamycin, N-benzyl-13-dihydroadriamycin, N,N-dibenzyldaunomycin, N,N-dibenzyl-13-dihydrodaunomycin and N,N-dibenzyladriamycin, and their pharmaceutically acceptable acid addition salts, together with a pharmaceutically acceptable nontoxic carrier or diluent therefor.
 10. A pharmaceutical composition of claim 9 wherein the dosage unit takes the form of a tablet.
 11. A pharmaceutical composition of claim 9 wherein the dosage unit takes the form of a capsule.
 12. A pharmaceutical composition of claim 9 wherein the dosage unit takes the form of a suppository.
 13. A pharmaceutical composition of claim 9 wherein the dosage unit takes the form of a cachet.
 14. A pharmaceutical composition in sterile aqueous form for treating leukemia L1210 and P388 in mice comprising an amount within the range from about 0.1 to about 500 mg, effective to ameliorate leukemia, of at least one compound selected from the group consisting of N-benzyldaunomycin, N-benzyl-13-dihydrodaunomycin, N-benzyladriamycin, N-benzyl-13-dihydroadriamycin, N,N-dibenzyldaunomycin, N,N-dibenzyl-13-dihydrodaunomycin and N,N-dibenzyladriamycin and their pharmaceutically acceptable acid addition salts, together with a pharmaceutically acceptable nontoxic, sterile, inert, aqueous carrier or diluent therefor.
 15. A pharmaceutical composition according to claim 14 in aqueous solution form. 